Modulation Of Neutrophil Migration Research Articles

Metformin Attenuates Neutrophil Recruitment through the H3K18 Lactylation/Reactive Oxygen Species Pathway in Zebrafish.

Beyond its well-established role in diabetes management, metformin has gained attention as a promising therapeutic for inflammation-related diseases, largely due to its antioxidant capabilities. However, the mechanistic underpinnings of this effect remain elusive. Using in vivo zebrafish models of inflammation, we explored the impact of metformin on neutrophil recruitment and the underlying mechanisms involved. Our data indicate that metformin reduces histone (H3K18) lactylation, leading to the decreased production of reactive oxygen species (ROS) and a muted neutrophil response to both caudal fin injury and otic vesicle inflammation. To investigate the precise mechanisms through which metformin modulates neutrophil migration via ROS and H3K18 lactylation, we meticulously established the correlation between metformin-induced suppression of H3K18 lactylation and ROS levels. Through supplementary experiments involving the restoration of lactate and ROS, our findings demonstrated that elevated levels of both lactate and ROS significantly promoted the inflammatory response in zebrafish. Collectively, our study illuminates previously unexplored avenues of metformin's antioxidant and anti-inflammatory actions through the downregulation of H3K18 lactylation and ROS production, highlighting the crucial role of epigenetic regulation in inflammation and pointing to metformin's potential in treating inflammation-associated conditions.

Cyclosporine A Modulates LSP1 Protein Levels in Human B Cells to Attenuate B Cell Migration at Low O2 Levels.

Coordinated migration of B cells within and between secondary lymphoid tissues is required for robust antibody responses to infection or vaccination. Secondary lymphoid tissues normally expose B cells to a low O (hypoxic) environment. Recently, we have shown that human B cell migration is modulated by an O-dependent molecular switch, centrally controlled by the hypoxia-induced (transcription) factor-1 (HIF1A), which can be disrupted by the immunosuppressive calcineurin inhibitor, cyclosporine A (CyA). However, the mechanisms by which low O environments attenuate B cell migration remain poorly defined. Proteomics analysis has linked CXCR4 chemokine receptor signaling to cytoskeletal rearrangement. We now hypothesize that the pathways linking the O sensing molecular switch to chemokine receptor signaling and cytoskeletal rearrangement would likely contain phosphorylation events, which are typically missed in traditional transcriptomic and/or proteomic analyses. Hence, we have performed a comprehensive phosphoproteomics analysis of human B cells treated with CyA after engagement of the chemokine receptor CXCR4 with CXCL12. Statistical analysis of the separate and synergistic effects of CyA and CXCL12 revealed 116 proteins whose abundance is driven by a synergistic interaction between CyA and CXCL12. Further, we used our previously described algorithm BONITA to reveal a critical role for Lymphocyte Specific Protein 1 (LSP1) in cytoskeletal rearrangement. LSP1 is known to modulate neutrophil migration. Validating these modeling results, we show experimentally that LSP1 levels in B cells increase with low O exposure, and CyA treatment results in decreased LSP1 protein levels. This correlates with the increased chemotactic activity observed after CyA treatment. Lastly, we directly link LSP1 levels to chemotactic capacity, as shRNA knock-down of LSP1 results in significantly increased B cell chemotaxis at low O levels. These results directly link CyA to LSP1-dependent cytoskeletal regulation, demonstrating a previously unrecognized mechanism by which CyA modulates human B cell migration. Data are available via ProteomeXchange with identifier PXD036167.

A curious case of cyclin-dependent kinases in neutrophils.

Neutrophils are terminally differentiated, short-lived white blood cells critical for innate immunity. Although cyclin-dependent kinases (CDKs) are typically related to cell cycle progression, increasing evidence has shown that they regulate essential functions of neutrophils. This review highlights the roles of CDKs and their partners, cyclins, in neutrophils, outside of cell cycle regulation. CDK1-10 and several cyclins are expressed in neutrophils, albeit at different levels. Observed phenotypes associated with specific inhibition or genetic loss of CDK2 indicate its role in modulating neutrophil migration. CDK4 and 6 regulate neutrophil extracellular traps (NETs) formation, while CDK5 regulates neutrophil degranulation. CDK7 and 9 are critical in neutrophil apoptosis, contributing to inflammation resolution. In addition to the CDKs that regulate mature neutrophil functions, cyclins are essential in hematopoiesis and granulopoiesis. The pivotal roles of CDKs in neutrophils present an untapped potential in targeting CDKs for treating neutrophil-dominant inflammatory diseases and understanding the regulation of the neutrophil life cycle.

SO079ASYMPTOMATIC HYPERURICEMIA, A REGULATOR OF INNATE IMMUNITY IN CHRONIC KIDNEY DISEASE

Abstract Background and Aims Asymptomatic hyperuricemia (HU) is common among patients with chronic kidney disease (CKD) but the causative role of HU on CKD progression remains controversial. Two large multi-center randomized controlled trials (RCTs), the CKD-FIX and the PERL study, have now disproven a causal relation. On the other hand, a causative role of persistent HU exists with gouty arthritis but a rapid correction of HU with urate lowering therapy can also elicit acute gout attacks. This suggests a more complex role of HU in this context. Hence, we hypothesized that soluble uric acid (sUA) has immunomodulatory effects on leukocyte function during the immune response to monosodium urate (MSU) crystals. Method Alb-creERT2;Glut9lox/lox and Glut9lox/lox control mice were injected with tamoxifen and placed either on a chow or high-fat diet with inosine to induce HU with or without CKD. After two weeks, MSU crystals or vehicle were injected into air pouches or postcapillary venules in the cremaster muscle of transgenic mice. Leukocyte infiltration and the extent of inflammation were assessed by flow cytometry, intravital microscopy, ELISA, and colorimetric assays. Human blood neutrophils and CD14+ monocytes were isolated from CKD stage G2-4 (n=10) and CKD stage G5 (n=18) patients or healthy individuals (n=15). Neutrophil transwell migration assays were performed and the number of migrated human neutrophils towards chemoattractants determined by flow cytometry. Human blood CD14+ monocytes were treated with MSU crystals or lipopolysaccharide ex vivo, and the expression of inflammatory mediators determined by RT-PCR and flow cytometry. Results We found that the presence of HU impaired leukocyte recruitment into MSU crystal-injected air pouches of mice with or without CKD. Intravital microscopy revealed that HU specifically reduced leukocyte adhesion, extravasation, and leukocyte-related tissue inflammation. The CKD-mediated attenuation of MSU crystal-induced inflammation was fully reversible by treating HU with urate lowering therapy such as rasburicase. In blood neutrophils isolated from healthy individuals, sUA diminished β2 integrin activation and expression, and hence impaired neutrophil migration ex vivo. An impaired migratory capability was also observed in neutrophils from CKD stage G2-4 or G5D patients. Moreover, sUA attenuated Toll-like and TNFα receptor-induced human monocyte activation, a process depending on the intracellular uptake of sUA via the urate transporter SLC2A9 (GLUT9), the only urate transporter expressed by these immune cells. Conclusion We identify sUA as an endogenous modulator of innate immunity. HU modulates neutrophil migration by altering efficient β2 integrin activation and suppresses pattern recognition receptor-driven monocyte activation via SLC2A9 in gouty arthritis related or unrelated to CKD (see Figure). This process provides a molecular explanation for several previously unexplained clinical phenomena in the context of gouty arthritis and renal failure.

Club cell protein 16 in sera from trauma patients modulates neutrophil migration and functionality via CXCR1 and CXCR2

BackgroundClub Cell protein (CC)16 correlates with lung injury and respiratory complications, which are in part triggered by polymorphonuclear leukocytes (PMNL) in severely traumatized patients (TP). CC16 exerts anti-inflammatory and immunosuppressive effects, however, its influence on PMNL functions after trauma is unknown. Here, we evaluated whether CC16 present in sera from TP could modify the biological functions of PMNL.MethodsSera from 16 severely injured TP without pneumonia (no P, n = 8) or with pneumonia (P, n = 8) were collected at admission to emergency department (ED) and 1 day prior pneumonia and pre-incubated with or without anti-CC16 antibody for CC16 neutralization. Samples from the equal post-injury days in the corresponding no P group were used. Neutrophils were isolated from healthy volunteers (HV, n = 5) and incubated with 20% of the serum medium from TP, respectively. In PMNL, CD62L, CD11b/CD18 and CD31 expression, migratory capacity, phagocytosis rate, oxidative burst and apoptosis were investigated. In isolated PMNL, CXCR1 and CXCR2 were neutralized before stimulation with CC16, and oxidative burst, phagocytosis and apoptosis were analyzed in neutrophils and their subsets.ResultsSerum from the P group enhanced significantly PMNL migration compared to no P group, while CC16-neutralization further increased the migratory rate of PMNL in both groups. CC16-neutralization increased significantly the expression of CD62L in the P group at ED. Oxidative burst was significantly increased in the P group vs. no P during the study period. CC16 seemed to have no influence on oxidative burst and phagocytosis in TP. However, in a more controlled study design, CC16 induced a significant increase of oxidative burst and a decrease of apoptosis of CD16+ granulocytes. These effects were markedly observed in mature CD16brightCD62Lbright and immune suppressive CD16brightCD62Ldim neutrophils. In mature subset, CXCR1 and CXCR2 neutralization diminished CC16-induced effects.ConclusionsCC16 in sera from multiply traumatized patients, notably of those with pneumonia, has significant effects on PMNL. The results suggest an association of CC16 with CXCR1 and CXCR2. Our data suggest that CC16 reduces the migratory capacity of PMNL and thus modulates their function in patients with respiratory complications after trauma.

Phenotypical microRNA screen reveals a noncanonical role of CDK2 in regulating neutrophil migration

Neutrophil migration is essential for inflammatory responses to kill pathogens; however, excessive neutrophilic inflammation also leads to tissue injury and adverse effects. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA (miRNA) overexpression screen in zebrafish and identified 8 miRNAs as potent suppressors of neutrophil migration. Among those, miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (Cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils without inducing cell death. Human neutrophil-like cells deficient in CDK2 fail to polarize and display altered signaling downstream of the formyl peptide receptor. Chemotaxis of primary human neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. Our results therefore reveal previously unknown functions of miR-199 and CDK2 in regulating neutrophil migration and provide directions in alleviating systemic inflammation.

The Alpha-Tocopherol Form of Vitamin E Boosts Elastase Activity of Human PMNs and Their Ability to Kill Streptococcus pneumoniae.

Despite the availability of vaccines, Streptococcus pneumoniae remains a leading cause of life-threatening infections, such as pneumonia, bacteremia and meningitis. Polymorphonuclear leukocytes (PMNs) are a key determinant of disease course, because optimal host defense requires an initial robust pulmonary PMN response to control bacterial numbers followed by modulation of this response later in infection. The elderly, who manifest a general decline in immune function and higher basal levels of inflammation, are at increased risk of developing pneumococcal pneumonia. Using an aged mouse infection model, we previously showed that oral supplementation with the alpha-tocopherol form of vitamin E (α-Toc) decreases pulmonary inflammation, in part by modulating neutrophil migration across lung epithelium into alveolar spaces, and reverses the age-associated decline in resistance to pneumococcal pneumonia. The objective of this study was to test the effect of α-Toc on the ability of neutrophils isolated from young (22–35 years) or elderly (65–69 years) individuals to migrate across epithelial cell monolayers in response to S. pneumoniae and to kill complement-opsonized pneumococci. We found that basal levels of pneumococcal-induced transepithelial migration by PMNs from young or elderly donors were indistinguishable, suggesting that the age-associated exacerbation of pulmonary inflammation is not due to intrinsic properties of PMNs of elderly individuals but rather may reflect the inflammatory milieu of the aged lung. Consistent with its anti-inflammatory activity, α-Toc treatment diminished PMN migration regardless of donor age. Unexpectedly, unlike previous studies showing poor killing of antibody-opsonized bacteria, we found that PMNs of elderly donors were more efficient at killing complement-opsonized bacteria ex vivo than their younger counterparts. We also found that the heightened antimicrobial activity in PMNs from older donors correlated with increased activity of neutrophil elastase, a serine protease that is required to kill pneumococci. Notably, incubation with α-Toc increased PMN elastase activity from young donors and boosted their ability to kill complement-opsonized pneumococci. These findings demonstrate that α-Toc is a potent modulator of PMN responses and is a potential nutritional intervention to combat pneumococcal infection.

Chemorepellent Semaphorin 3E Negatively Regulates Neutrophil Migration In Vitro and In Vivo.

Neutrophil migration is an essential step in leukocyte trafficking during inflammatory responses. Semaphorins, originally discovered as axon guidance cues in neural development, have been shown to regulate cell migration beyond the nervous system. However, the potential contribution of semaphorins in the regulation of neutrophil migration is not well understood. This study examines the possible role of a secreted chemorepellent, Semaphorin 3E (Sema3E), in neutrophil migration. In this study, we demonstrated that human neutrophils constitutively express Sema3E high-affinity receptor, PlexinD1. Sema3E displayed a potent ability to inhibit CXCL8/IL-8-induced neutrophil migration as determined using a microfluidic device coupled to real-time microscopy and a transwell system in vitro. The antimigratory effect of Sema3E on human neutrophil migration was associated with suppression of CXCL8/IL-8-mediated Ras-related C3 botulinum toxin substrate 1 GTPase activity and actin polymerization. We further addressed the regulatory role of Sema3E in the regulation of neutrophil migration in vivo. Allergen airway exposure induced higher neutrophil recruitment into the lungs of Sema3e-/- mice compared with wild-type controls. Administration of exogenous recombinant Sema3E markedly reduced allergen-induced neutrophil recruitment into the lungs, which was associated with alleviation of allergic airway inflammation and improvement of lung function. Our data suggest that Sema3E could be considered an essential regulatory mediator involved in modulation of neutrophil migration throughout the course of neutrophilic inflammation.

G Protein-Coupled Receptor 43 Modulates Neutrophil Recruitment during Acute Inflammation.

Fermentation of dietary fibre in the gut yields large amounts of short chain fatty acids (SCFAs). SCFAs can impart biological responses in cells through their engagement of ‘metabolite-sensing’ G protein-coupled receptors (GPCRs). One of the main SCFA receptors, GPR43, is highly expressed by neutrophils, which suggests that the actions of GPR43 and dietary fibre intake may affect neutrophil recruitment during inflammatory responses in vivo. Using intravital imaging of the small intestine, we found greater intravascular neutrophil rolling and adhesion in Gpr43−/−mice in response to LPS at 1 h. After 4 h of LPS challenge, the intravascular rolling velocity of GPR43-deficient neutrophils was reduced significantly and increased numbers of neutrophils were found in the lamina propria of Gpr43−/−mice. Additionally, GPR43-deficient leukocytes demonstrated exacerbated migration into the peritoneal cavity following fMLP challenge. The fMLP-induced neutrophil migration was significantly suppressed in wildtype mice that were treated with acetate, but not in Gpr43−/−mice, strongly suggesting a role for SCFAs in modulating neutrophil migration via GPR43. Indeed, neutrophils of no fibre-fed wildtype mice exhibited elevated migratory behaviour compared to normal chow-fed wildtype mice. Interestingly, this elevated migration could also be reproduced through simple transfer of a no fibre microbiota into germ-free mice, suggesting that the composition and function of microbiota stemming from a no fibre diet mediated the changes in neutrophil migration. Therefore, GPR43 and a microbiota composition that allows for SCFA production function to modulate neutrophil recruitment during inflammatory responses.

Vestitol Isolated from Brazilian Red Propolis Inhibits Neutrophils Migration in the Inflammatory Process: Elucidation of the Mechanism of Action.

Vestitol is an isoflavonoid isolated from Brazilian red propolis with potential anti-inflammatory activity. This study investigated the mechanism of action of vestitol on the modulation of neutrophil migration in the inflammatory process. Pre-treatment with vestitol at 1, 3, or 10 mg/kg reduced LPS- or mBSA-induced neutrophil migration and the release of CXCL1/KC and CXCL2/MIP-2 in vivo. Likewise, pre-treatment with vestitol at 1, 3, or 10 μM reduced the levels of CXCL1/KC and CXCL2/MIP-2 in macrophage supernatants in vitro. Moreover, the administration of vestitol (10 mg/kg) reduced leukocyte rolling and adherence in the mesenteric microcirculation of mice. The pre-treatment with vestitol (10 mg/kg) in iNOS(-/-) mice did not block its activity concerning neutrophil migration. With regard to the activity of vestitol on neutrophils isolated from the bone marrow of mice, there was a reduction on the chemotaxis of CXCL2/MIP-2 or LTB4-induced neutrophils and on calcium influx after pre-treatment with the compound at 3 or 10 μM. There was no change in CXCR2 expression by neutrophils treated with vestitol at 10 μM. These findings demonstrate that vestitol is a promising novel anti-inflammatory agent.

Riparin A, a compound from Aniba riparia, attenuate the inflammatory response by modulation of neutrophil migration

Inflammation is a local tissue response to attacks characterized by vascular and cellular events, including intense oxidative stress. Riparin A, a compound obtained from Aniba riparia, has been shown to have antioxidant activity and cytotoxicity in vitro. This study was aimed at evaluating the anti-inflammatory effect of riparin A against acute inflammation. The results of our evaluations in various experimental models indicated that riparin A reduced paw edema induced by carrageenan, compound 48/80, histamine, and serotonin. Furthermore, it decreased leukocyte and neutrophil counts, myeloperoxidase activity, thiobarbituric acid reactive substance (TBARS) levels, and cytokine (tumor necrosis factor-α and interleukin-1β) levels increased by carrageenan-induced peritonitis, and reversed glutathione levels. Riparin A also reduced carrageenan-induced adhesion and rolling of leukocytes on epithelial cells and did not produce gastric-damage as compared with indomethacin. In conclusion, the data show that riparin A reduces inflammatory response by inhibiting vascular and cellular events, modulating neutrophil migration, inhibiting proinflammatory cytokine production, and reducing oxidative stress.

Blockade of ICAM-1 improves the outcome of polymicrobial sepsis via modulating neutrophil migration and reversing immunosuppression.

Intercellular adhesion molecule-1 (ICAM-1) is a key adhesion molecule mediating neutrophil migration and infiltration during sepsis. But its role in the outcome of sepsis remains contradictory. The current study was performed to investigate the role of anti-ICAM-1 antibody in the outcome of polymicrobial sepsis and sepsis-induced immune disturbance. Effect of anti-ICAM-1 antibody on outcome of sepsis induced by cecal ligation and puncture (CLP) was evaluated by the survival analysis, bacterial clearance, and lung injury. Its influence on neutrophil migration and infiltration, as well as lymphocyte status, in thymus and spleen was also investigated. The results demonstrated that ICAM-1 mRNA was upregulated in lung, thymus, and spleen of CLP mice. Anti-ICAM-1 antibody improved survival and bacterial clearance in CLP mice and attenuated lung injury. Migration of neutrophils to peritoneal cavity was enhanced while their infiltration into lung, thymus, and spleen was hampered by ICAM-1 blockade. Anti-ICAM-1 antibody also prevented sepsis-induced apoptosis in thymus and spleen. Positive costimulatory molecules including CD28, CD80, and CD86 were upregulated, while negative costimulatory molecules including PD-1 and PD-L1 were downregulated following anti-ICAM-1 antibody administration. In conclusion, ICAM-1 blockade may improve outcome of sepsis. The rationale may include the modulated neutrophil migration and the reversed immunosuppression.

Amitriptyline, clomipramine, and maprotiline attenuate the inflammatory response by inhibiting neutrophil migration and mast cell degranulation

Despite the recognized anti-inflammatory potential of heterocyclic antidepressants, the mechanisms concerning their modulating effects are not completely known. Thus, we evaluated the anti-inflammatory effect of amitriptyline, clomipramine, and maprotiline and the possible modulating properties of these drugs on neutrophil migration and mast cell degranulation. The hind paw edema and air-pouch models of inflammation were used. Male Wistar rats were treated with saline, amitriptyline, clomipramine or maprotiline (10, 30, or 90 mg/kg, per os [p.o.]) 1 h before the injection of carrageenan (300 μg/0.1 mL/paw) or dextran (500 μg/0.1 mL/paw). Then, edema formation was measured hourly. Neutrophil migration to carrageenan (500 μg/pouch) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10-6 M/mL/pouch) was also investigated in 6-day-old air-pouch cavities. Compound 48/80-induced mast cell degranulation was assessed in the mesenteric tissues of antidepressant-treated rats. All tested antidepressants prevented both carrageenan- and dextran-induced edema. The anti-inflammatory effect of these drugs partially depends on the modulation of neutrophil migration, since they significantly counteracted the chemotactic response of both carrageenan and fMLP (p < 0.01). Furthermore, amitriptyline, clomipramine and maprotiline inhibited compound 48/80-induced mast cell degranulation (p < 0.001). These results suggest an important anti-inflammatory role of heterocyclic antidepressants, which is dependent on the modulation of neutrophil migration and mast cell stabilization.

Modulation of the inflammatory response to enterohemorrhagic E. coli by the secreted mucinase StcE

Enterohemorrhagic E. coli (EHEC) are human bacterial pathogens that have caused numerous outbreaks of foodborne illness worldwide. EHEC infection causes hemorrhagic colitis that in 5–10% of cases can progress to the potentially fatal hemolytic uremic syndrome (HUS). Although EHEC is not considered a classical inflammatory pathogen, clinical data suggest that inflammation may play a role during intestinal infection and in kidney damage during HUS. Our aim is to understand the molecular mechanisms used by EHEC to elicit a pathologic host immune response. Using our well‐established in vitro model of transepithelial migration of neutrophils, we found that EHEC induce migration of neutrophils across polarized epithelial monolayers. We further demonstrated that secretion of StcE during EHEC infection promotes neutrophil transepithelial migration. StcE is a large zinc metalloprotease, involved in cleavage of the mucin‐type O‐glycosylated proteins CD43 and CD45 at the neutrophil surface. CD43 provides an antiadhesive force in cell‐cell interactions and regulates leukocyte adhesion and migration. Treatment with purified StcE rendered neutrophils more adherent in vitro. Our results suggest that during EHEC infection, modulation of neutrophil migration by StcE may play a pro‐inflammatory role that contributes to a dysregulated immune response and the subsequent development of HUS.

Anti-inflammatory effect of a sulphated polysaccharide fraction extracted from the red algaeHypnea musciformisvia the suppression of neutrophil migration by the nitric oxide signalling pathway

The aim of this study was to evaluate the anti-inflammatory effect of a sulphated polysaccharide fraction (PLS) extracted from the alga Hypnea musciformis and investigate the possible involvement of the nitric oxide (NO) pathway in this effect. The anti-inflammatory activity of PLS was evaluated using inflammatory agents (carrageenan and dextran) to induce paw oedema and peritonitis in Swiss mice. Samples of paw tissue and peritoneal fluid were removed to determine myeloperoxidase (MPO) activity, NO3 /NO2 levels, and interleukin-1β (IL-1β) level. The involvement of NO in the modulation of neutrophil migration in carrageenan-induced paw oedema or peritonitis was also investigated. Compared with vehicle-treated mice, mice pretreated with PLS (10 mg/kg) inhibited carrageenan-induced and dextran-induced oedema; it also inhibited total and differential peritoneal leucocyte counts in a model of peritonitis. These PLS effects were reversed by l-arginine treatment and recovered with the administration of a NO synthase blocker (aminoguanidine). Furthermore, PLS reduced the MPO activity, decreased IL-1β levels, and increased NO3 /NO2 levels in the peritoneal cavity. PLS reduced the inflammatory response by modulating neutrophil migration, which appeared to be dependent on the NO pathway.

Bioactive Fraction of Geopropolis fromMelipona scutellarisDecreases Neutrophils Migration in the Inflammatory Process: Involvement of Nitric Oxide Pathway

The aim of this study was to evaluate the activity of the ethanolic extract of geopropolis (EEGP) from Melipona scutellaris and its fractions on the modulation of neutrophil migration in the inflammatory process, and the participation of nitric oxide (NO) pathway, as well as to check the chemical profile of the bioactive fraction. EEGP and its aqueous fraction decreased neutrophil migration in the peritoneal cavity and also the interaction of leukocytes (rolling and adhesion) with endothelial cells. The levels of chemokines CXCL1/KC and CXCL2/MIP-2 were not altered after treatment with EEGP and the aqueous fraction. It was found that the injection of NO pathway antagonists abolished the EEGP and the aqueous fraction inhibitory activity on the neutrophil migration. The expression of intercellular adhesion molecule type 1 (ICAM-1) was reduced, and nitrite levels increased after treatment with EEGP and aqueous fraction. In the carrageenan-induced paw edema model, EEGP and the aqueous fraction showed antiedema activity. No pattern of flavonoid and phenolic acid commonly found in propolis samples of Apis mellifera could be detected in the aqueous fraction samples. These data indicate that the aqueous fraction found has promising bioactive substances with anti-inflammatory activity.

WFDC1/ps20: A host factor that influences the neutrophil response to murine hepatitis virus (MHV) 1 infection

The whey acidic protein family member, WFDC1/ps20 is a permissivity factor in HIV infection. Herein we describe a contrasting role for ps20 in limiting MHV-1 infection. Intranasal MHV-1 infection produces a respiratory infection in mice. Using ps20 knockout mice we provide evidence that intranasal MHV-1 infection results in increased lung viral titers in ps20−/− compared to ps20+/+ mice. Accompanying MHV-1 infection we observe an increase in the number of neutrophils infiltrating the BAL and an increase in the percentage of neutrophils in the lung draining lymph nodes of ps20−/− compared with ps20+/+ mice. Gene expression levels for the neutrophil chemoattractants CXCL1 and CXCL2 are elevated in the lungs of ps20−/− mice post-MHV-1 infection. Characterization of the immune cell profile in naïve ps20−/− mice revealed an increase in circulating neutrophils compared to ps20+/+ mice. No notable differences in other immune cell profiles were observed between the ps20+/+ and ps20−/− mice. Accordingly, we examined MHV-1 infection of neutrophils and provide evidence that neutrophils isolated from ps20−/− mice are more susceptible to MHV-1 infection than neutrophils isolated from ps20+/+ mice. These data suggest roles for ps20 in regulating expression of neutrophil-specific chemotactic factors, thereby potentially modulating neutrophil migration, and in modulating neutrophil susceptibility to MHV-1 infection.

Hydrogen Sulfide Augments Neutrophil Migration through Enhancement of Adhesion Molecule Expression and Prevention of CXCR2 Internalization: Role of ATP-Sensitive Potassium Channels

In this study, we have addressed the role of H(2)S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H(2)S synthesis inhibitors, dl-propargylglycine (PAG) or beta-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H(2)S donors, NaHS or Lawesson's reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-alpha, keratinocyte-derived chemokine, and LTB(4). Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (K(ATP)(+)) channel blocker, glybenclamide. Conversely, diazoxide, a K(ATP)(+) channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H(2)S augments neutrophil adhesion and locomotion, by a mechanism dependent on K(ATP)(+) channels.

Activation of normal neutrophils by anti-neutrophil cytoplasm antibodies.

Anti-neutrophil cytoplasm antibodies (ANCA) are markers of systemic vasculitis for which a pathogenetic role has been postulated. We have examined the effect of these autoantibodies on the function of normal human neutrophils in vitro. In the presence of ANCA positive sera luminol-amplified chemiluminescence was significantly increased compared to the values seen in the presence of normal or anti-double stranded DNA positive sera (P < 0.01). Five of six ANCA positive F(ab)2 preparations also produced significant neutrophil activation as demonstrated by the chemiluminescence response. This response was totally abrogated by the addition of neutrophil cytoplasm extract, containing the ANCA antigen. Addition of inhibitors to the chemiluminescence system demonstrated that the chemiluminescence response was inhibited by azide and salicylhydroxamic acid and reduced by histidine, suggesting that the chemiluminescence response was due to activation of myeloperoxidase, with generation of singlet oxygen. The chemotactic response to f-Met-Leu-Phe, a bacterial chemotactic peptide, was significantly augmented in the presence of ANCA. Chemotaxis to zymosan-activated serum and chemokinesis was not affected. Phagocytosis was also unaffected. We propose that neutrophil activation and modulation of neutrophil migration by ANCA may be of pathogenetic significance in systemic vasculitis.

Nitric Oxide Regulates Neutrophil Migration through Microparticle Formation

The role of nitric oxide (NO) in regulating neutrophil migration has been investigated. Human neutrophil migration to interleukin (IL)-8 (1 nmol/L) was measured after a 1-hour incubation using a 96-well chemotaxis plate assay. The NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) significantly (P < 0.001) enhanced IL-8-induced migration by up to 45%. Anti-CD18 significantly (P < 0.001) inhibited both IL-8-induced and L-NAME enhanced migration. Antibodies to L-selectin or PSGL-1 had no effect on IL-8-induced migration but prevented the increased migration to IL-8 induced by L-NAME. L-NAME induced generation of neutrophil-derived microparticles that was significantly (P < 0.01) greater than untreated neutrophils or D-NAME. This microparticle formation was dependent on calpain activity and superoxide production. Only microparticles from L-NAME and not untreated or D-NAME-treated neutrophils induced a significant (P < 0.01) increase in IL-8-induced migration and transendothelial migration. Pretreatment of microparticles with antibodies to L-selectin (DREG-200) or PSGL-1 (PL-1) significantly (P < 0.001) inhibited this effect. The ability of L-NAME-induced microparticles to enhance migration was found to be dependent on the number of microparticles produced and not an increase in microparticle surface L-selectin or PSGL-1 expression. These data show that NO can modulate neutrophil migration by regulating microparticle formation.